Chitosan Scaffolds as Microcarriers for Dynamic Culture of Human Neural Stem Cells

Abstract

Human neural stem cells (hNSCs) possess remarkable potential for regenerative medicine in the treatment of presently incurable diseases. However, a key challenge lies in producing sufficient quantities of hNSCs, which is necessary for effective treatment. Dynamic culture systems are recognized as a powerful approach to producing large quantities of hNSCs required, where microcarriers play a critical role in supporting cell expansion. Nevertheless, the currently available microcarriers have limitations, including a lack of appropriate surface chemistry to promote cell adhesion, inadequate mechanical properties to protect cells from dynamic forces, and poor suitability for mass production. Here, we present the development of three-dimensional (3D) chitosan scaffolds as microcarriers for hNSC expansion under defined conditions in bioreactors. We demonstrate that chitosan scaffolds with a concentration of 4 wt% (4CS scaffolds) exhibit desirable microstructural characteristics and mechanical properties suited for hNSC expansion. Furthermore, they could also withstand degradation in dynamic conditions. The 4CS scaffold condition yields optimal metabolic activity, cell adhesion, and protein expression, enabling sustained hNSC expansion for up to three weeks in a dynamic culture. Our study introduces an effective microcarrier approach for prolonged expansion of hNSCs, which has the potential for mass production in a three-dimensional setting.

A Chitosan Scaffold Supports the Enhanced and Prolonged Differentiation of HiPSCs into Nucleus Pulposus-like Cells

“…To ensure homogeneity and the removal of air bubbles, the solution was mixed again in the Thinky mixer. The resulting solution was cast into commercial tissue culture microplates, refrigerated at 4 °C for 1 h, at −5 °C for 20 min, at −20 °C for 1 h, and then anneal at −2 °C for 2 h, followed by lyophilization using a SP VirTis Genesis Pilot lyophilizer (SP Scientific, USA) . Prior to cell seeding, the chitosan scaffolds were sectioned into 400 μm slices and disinfected with 70 and 90% ethanol, followed by rinsing with PBS 3 times for 10 min each.…”

“…Prior studies have indicated that chitosan scaffolds with near-neutral charge enhance the adhesion and proliferation of human neural stem cells, outperforming negatively charged collagen scaffolds and chitosan−hyaluronic hybrid scaffolds. 39,40 Additionally, it has been reported that pure chitosan materials enhance the proliferation and pluripotency of hiPSCs. 31,41 Here, the zeta potential of our chitosan scaffolds showed minimal variation, maintaining nearneutrality despite changes in pH, which suggests a favorable setting for iPSCs attachment.…”

“…Chitosan is a naturally derived polysaccharide with intrinsic advantages for tissue engineering, such as its biocompatibility and structural congruence with native extracellular matrix (ECM) components including glycosaminoglycans and collagen . The chitosan-based 3D culture platform has proven highly effective in supporting the growth, renewal, and expansion of both human induced pluripotent stem cells (hiPSCs) and human neural stem cells. , Additionally, their ease of processing and cost-effectiveness make chitosan scaffolds uniquely well-suited as a promising platform for stem cell differentiation. We engineer chitosan scaffolds with optimal stiffness to replicate the natural environment of a healthy nucleus pulposus, ensuring sufficient mechanical strength for potential biomaterial implantation.…”

Advances in the application of extracellular vesicles derived from three-dimensional culture of stem cells

“…These scaffolds can be classified into three types: natural scaffolds, synthetic scaffolds and hybrid scaffolds [ 98 ]. Natural scaffolds mainly include scaffolds made of natural biomaterials such as collagen [ 99 ], fibrin [ 100 , 101 ], gelatin [ 102 , 103 ], hyaluronic acid [ 104 ], chitosan [ 105 ], alginate [ 106 ], agarose [ 107 ], matrigel [ 108 ] and other biomaterials. On the other hand, synthetic scaffolds contain scaffolds synthesized from materials such as polyethylene glycol (PEG) [ 109 , 110 ], polyvinyl alcohol (PVA) [ 111 ], poly lactic-co-glycolic acid (PLGA) [ 112 , 113 ], polylactic acid (PLA) [ 114 ], polycaprolactone (PCL) [ 115 , 116 ] and others [ 70 ].…”

“…On the other hand, synthetic scaffolds contain scaffolds synthesized from materials such as polyethylene glycol (PEG) [ 109 , 110 ], polyvinyl alcohol (PVA) [ 111 ], poly lactic-co-glycolic acid (PLGA) [ 112 , 113 ], polylactic acid (PLA) [ 114 ], polycaprolactone (PCL) [ 115 , 116 ] and others [ 70 ]. Both natural and synthetic materials can be fabricated into porous scaffolds for 3D cell culture [ 105 , 112 ]. While natural scaffolds offer better biocompatibility, they may vary between batches or donors.…”